Background
Acute myeloid leukemia (AML) is the most common acute leukemia, with treatment outcomes remaining poor. FMS-like tyrosine kinase 3 (FLT3) is highly expressed in over 80% of AML cases, on both AML blasts and leukemic stem cells, while its expression on hematopoietic stem cells was low. CD3 T cell engaging bispecific antibodies (TCE) redirect T cells to attack targeted tumor cells by simultaneously binding TAA on cancer cells and CD3 complex on a T-cell to form a TCR-independent immune synapse. Multiple TCEs have demonstrated promising therapeutic efficacy in the treatment of hematological malignancies. Here we developed a novel 2+1 FLT3 x CD3 TCE, ATG-107, based on the AnTenGager TM platform. It binds to the extracellular domain of FLT3 bivalently, inducing T cell-dependent cellular cytotoxicity, leading to a potent in vitro and in vivo preclinical efficacy.
Methods
ATG-107 was constructed by introducing a high affinity, novel conformational epitope-targeted anti-CD3 single chain fragment variable (scFv) antibody to the hinge region of one of the heavy chains of an anti-FLT3 monoclonal antibody. The CD3 binding site is concealed by the FLT3 Fab arm before binding to FLT3, due to the steric hindrance. ATG-107 was evaluated in a series of preclinical studies for binding affinity, T cell activation, T cell dependent cytotoxicity (TDCC). The in vivo antitumor efficacy of ATG-107 was evaluated in a humanized PBMCs and MV4-11-luciferase cells (+/ITD) engrafted B-NDG mice model.
Results
ATG-107 bound to FLT3 positive cells with a sub-nM EC50 and showed limited binding capability to CD3+ cells before FLT3 crosslinking. With the presence of FLT3 positive EOL-1 (+/+), THP-1 cell (+/+), MOLM-13(+/ITD) and MV4-11 (ITD/ITD), ATG-107 strongly activated primary T cells, upregulating early and later surface markers of T cell activation, such as CD25 and CD69. ATG-107 induced strong TDCC against FLT3-high/medium/low expressing AML cells regardless of the genetic alternation status of FLT3 (figure 1). In addition, ATG-107 demonstrated more potent in vivo anti-AML efficacy compared to the clinical benchmark antibody at 1 mg/kg dose level in PBMC-humanized B-NDG mice bearing MV4-11-luciferase AML cells.
Conclusions
ATG-107 demonstrates FLT3-dependent T cell activation, leading to potent preclinical efficacy. It may be a promising strategy for the treatment of a broader AML patient population.
Abstract 1068 Figure 1
ATG-107 shows significant antitumor effects in vitro and in vivo. A. Structural characteristics of BMK (benchmark) (left) and ATG-107 (right). BMK is an analog of CLN-049, produced using sequences from the patent WO2020053300A1. B. FACS binding analysis of BMK and ATG-107 to human FLT3-expressing HEK293F cells C. FACs binding analysis of BMK and ATG-107 to Jurkat cells (CD3+). Surface copy number, genetic status and expression of FLT3 on EOL-1 (D), THP-1 (E), MOLM-13 (F) and MV4-11 (G) cells. WT, wildtype; ITD, Internal tandem duplication. Antibody induced T cell activation (middle) and T cell dependent cellular cytotoxicity (TDCC, bottom) in presence of EOL-1, THP-1, MOLM-13 and MV4-11 were also shown in D, E, F and G, respectively